Inhibiting or genetically deleting the enzyme MTHFD2 reduce disease severity in multiple inflammatory disease


A metabolic enzyme that has been studied in cancer biology and is important for T cell function may offer a new target for anti-inflammatory therapeutics, Vanderbilt researchers have discovered.

They report Nov. 11 in the journal Immunity that inhibiting or genetically deleting the enzyme, called MTHFD2, reduced disease severity in multiple inflammatory disease models.

Jeffrey Rathmell, Ph.D., Cornelius Vanderbilt Professor of Immunobiology, and his team are interested in how metabolic pathways – the chemical reactions that sustain life – influence immune cell function. In the current studies, they focused on “one-carbon” metabolism, a series of reactions that generates chemical building blocks for the biosynthesis of DNA and other molecules.

“One-carbon metabolism has been a target for drug development for years and years, but it really hasn’t been explored in an unbiased way,” said Rathmell, who is also director of the Vanderbilt Center for Immunobiology. The immunosuppressant drug methotrexate, for example, inhibits an enzyme in the one-carbon metabolism pathway, but it may not be the “right target or the right drug” for optimal therapeutic activity, he said.

To systematically study the pathway in T cells – white blood cells that respond to specific antigens (such as surface proteins on viruses) – Ayaka Sugiura, an MD-Ph.D. student in Rathmell’s group, developed a screening strategy using the genome editing technology CRISPR.

She designed CRISPR “guides” to selectively inactivate each gene in the one-carbon metabolism pathway and introduced this “library” into isolated T cells, carefully controlling the experimental conditions so that each cell had only one (or no) inactivated gene.

By studying the modified cells in an animal model of asthma, Sugiura was able to identify genes important to T cell function during the disease process. She then examined the expression of each identified gene during T cell development and in patients with a variety of inflammatory diseases.

MTHFD2 stood out. It was highly expressed in disease states and during embryonic development, but it was expressed at low levels, or not at all, in adult tissues, Sugiura said.

MTHFD2 had previously been a target for anti-cancer drug development because of its overexpression in many tumors. Although preclinical studies did not support further anti-cancer development of MTHFD2 inhibitors, Sugiura was able to use a well-characterized inhibitor in her studies.

“MTHFD2 is important for nucleotide synthesis not only for DNA, but also for proper signaling required for T cell function,” Sugiura said. Inhibiting MTHFD2 with a drug or genetically eliminating it reduced overall proliferation of CD4 T cells (a particular type of T cell the group studied) and blunted immune responses, she said.

The researchers discovered, however, that the effects of MTHFD2 inhibition were different for subsets of CD4 T cells that are generated in response to antigen stimulation.

Inhibiting MTHFD2 promoted the activity of regulatory CD4 T cells (Treg), which suppress the immune response. But inhibiting MTHFD2 blocked inflammatory CD4 T cells (Th17) and actually converted them to an anti-inflammatory phenotype.

“This was pretty surprising,” Rathmell said. “Ayaka was able to show that inhibiting MTHFD2 doesn’t just stop an immune response, it actually switches it from inflammatory to anti-inflammatory.”

In animal models for multiple sclerosis, inflammatory bowel disease, and a general allergic response, inhibiting or eliminating MTHFD2 reduced disease severity, supporting its potential as a therapeutic target for anti-inflammatory drug development. The Rathmell group is working with collaborators to develop inhibitors with improved clinical characteristics.

The researchers also were encouraged to find that giving an MTHFD2 inhibitor in a vaccination model did not impair the immune response to a vaccine.

“It was promising that while the inhibitor suppressed inflammation in multiple disease models of hyperactive T cell activity, it did not affect desirable T cell responses, such as the response to vaccination,” Sugiura said.

The findings suggest that immune cell subsets rely on one-carbon metabolism – and MTHFD2 function – in different ways, the researchers noted.

And although MTHFD2 inhibitors were not successful as anti-cancer agents in general, they might be useful for cancers driven by inflammation, such as colorectal cancer. An MTHFD2 inhibitor would be expected to slow down cancer cell proliferation and also block “the specific inflammatory T cells that can promote that type of cancer,” Rathmell said.

The Rathmell group is using the CRISPR-based screen Sugiura developed to explore multiple sets of genes in various disease models and is working to build a core resource for other Vanderbilt investigators.

“This screening strategy and whole approach to look for important disease genes, which might be therapeutic targets, in an unbiased way is really valuable and has been very impactful for our group,” Rathmell said.

MTHFD2 is a bifunctional NMDMC. Its activity in transformed and non-differentiated cells was firstly detected in 1985 (7) and the cDNA cloning human MTHFD2 was isolated in 1989 (8). MTHFD2 had been reported to participate in the production of formyltetrahydrofolate for the synthesis of formylmethionyl transfer RNA required for the initiation of protein synthesis (9). The MTHFD2 protein had long been thought to be located exclusively in mitochondria until recently when it was also found to be present within the nucleus at the site of newly synthesized DNA (10).

The canonical role of MTHFD2 is central to folate-mediated one-carbon metabolism in mitochondria. A one-carbon unit (1C) from serine is transferred to tetrahydrofolate (THF) by serine hydroxymethyl transferases (SHMTs) to form 5,10-methylenetetrahydrofolate (methylene-THF/CH2-THF). The 1C unit is then transferred among different forms of THFs, thus enabling the folate cycle (Figure 1).

This biochemical network comprises two parallel metabolic reactions that take place in the cytoplasmic and mitochondrial compartments. In the cytoplasm, a single trifunctional enzyme named MTHFD1 comprises all the three domains (methylenetetrahydrofolate dehydrogenase, cyclohydrolase, and formyltetrahydrofolate synthetase domains), and serves as the primary functional enzyme that interconverts CH2-THF to 10-formyl-tetrahydrofolate (10-formyl-THF/10-CHO-THF).

In the mitochondria, the reactions are carried out by two MTHFD isozymes, MTHFD2 and MTHFD2L (11). They catalyze the production of 10-CHO-THF via two steps. The first one is the conversion of CH2-THF to 5,10-methenyl-tetrahydrofolate (methenyl-THF/CH+-THF) through the dehydrogenase activity, the second step is the conversion of CH+-THF to 10-CHO-THF by the cyclohydrolase domain (12) (Figure 1).

MTHFD2 has been recognized to use NAD+ as a cofactor in the oxidation process while MTHFD2L can use both NAD+ and NADP+. A recent report, however, demonstrated that MTHFD2 can also use both NAD+ and NADP+ in rapidly proliferating cells (13), suggesting an additional uncharacterized antioxidative role. Compared with MTHFD2L isozyme, MTHFD2 was reported to have much higher expression (14) and displayed a more predominant role in maintaining mitochondrial folate pathway function as well as responding to growth factor stimulation (15). Thus, therapeutic strategies targeting the mitochondrial folate pathway could be simplified by specifically focusing on MTHFD2.

Figure 1. Folate one-carbon metabolism. One carbon metabolism enzymes are presented and activated in three different compartments, the nucleus, cytosol, and mitochondria. The flow of permeable metabolites linked the reactions between these compartments, such as formate, serine, and glycine. Briefly, different forms of THFs, function as carriers, transfer one-carbon units as from serine to formate in the mitochondria. Formate then supply the biosynthesis of purine in the cytosol and thymidylate in the nucleus. The reactions in mitochondria are catalyzed mainly by SHMT2, MTHFD2, and MTHFD1L; while in cytosol and nucleus are by SHMT1/2 and MTHFD1. The enzymatic functions of MTHFD2 in the mitochondria are well-studied, while its role in the nucleus is largely unknown and may hold various non-metabolic functions.

MTHFD2 (350 amino acids, 37kDa) is one of the major enzymes involved in mitochondrial folate one-carbon metabolism and is also known as NMDMC (NAD-dependent mitochondrial methylenetetrahydrofolate dehydrogenase-cyclohydrolase). Despite its well-known bifunctional dehydrogenase and cyclohydrolase activities, MTHFD2 has been reported to be required for cancer proliferation and may have profound role in tumor development and progression.

This metabolic enzyme has attracted particular interests in cancer research for several reasons.

Firstly, MTHFD2 is upregulated in various cancers, transformed cells, and developing embryos, but has low or undetectable level in most differentiated normal adult tissues (1).

Secondly, highly expressed MTHFD2 is associated with poor disease outcomes in breast cancer (2), colorectal cancer (CRC) (3), renal cell carcinoma (RCC) (4), and hepatocellular carcinoma (HCC) (5); upregulation of MTHFD2 may also contribute to an increased risk of bladder cancer (6).

Thirdly, depletion of MTHFD2 may impair aggressive phenotypes and cause cell death in multiple cancers (1). Taken together, MTHFD2 is oncogenic in nature and may serve as a prognostic indicator as well as a therapeutic target in cancers.

Yet, the physiological role of MTHFD2 in malignancy and the mechanisms contributing to its pro-oncogenic activities have not yet been fully elucidated. A better understanding of both the enzymatic and non-enzymatic functional roles of MTHFD2 is essential for the optimal targeting of this novel candidate in cancer therapy.

This review aims to highlight the potential functions of MTHFD2 in cancers, particularly focusing on its diagnostic/prognostic value and the effects of its knockdown on aggressive phenotypes. We will summarize the regulatory mechanisms of MTHFD2 and the effects after its depletion, including cell morphological changes, oxidative homeostasis, and metabolite profile alterations. The non-enzymatic “moonlighting” function of MTHFD2 and the development of MTHFD2 inhibitors will also be discussed.

reference link :

More information: Jeffrey C. Rathmell, MTHFD2 is a Metabolic Checkpoint Controlling Effector and Regulatory T Cell Fate and Function, Immunity (2021). DOI: 10.1016/ … 1074-7613(21)00448-9


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